G protein coupled receptors (GPCRs) are crucial for the transduction of extracellular stimuli to the intracellular space. Upon activation, GPCRs undergo large conformational changes to engage transducers and stimulate intracellular responses. However, the kinetics of agonist induced GPCR conformational changes are relatively understudied. An exception to this is the class A rhodopsin receptor, which has a covalent agonist and fast (< 1ms) activation kinetics. In contrast, other GPCRs are thought to activate across the low to mid millisecond range [1]. For Class C GPCRs, which are distinct from class A receptors in that they contain large extracellular agonist binding domains and exist as obligate dimers, the site of agonist binding is >100Å from where the transducer interacts [2]. Class C GPCR activation involves both dimer rearrangement and activation of the 7-transmembrane (7-TM) domain, which are thought to occur over 20-200ms [3-5]. An outstanding question is whether the activation kinetics of rhodopsin are indeed faster than other GPCRs, or if previous experimental approaches lacked sufficient resolution to reveal fast kinetics in other receptor families.
To this end, Grushevskyi and colleagues have used FRET recordings to detect submillisecond activation dynamics of a prototypical class C GPCR, metabotropic glutamate receptor subtype 1 (mGlu1), demonstrating that mGlu1 undergoes two temporally distinct conformational changes upon activation [6]. Inter-subunit movements were detected by labelling the second intracellular loop of one protomer with CFP and the other with YFP. Intra-subunit changes detected by labelling each protomer with YFP in the second intracellular loop and CFP in the C-terminus. Synchronous activation of receptors was achieved via two complementary methods. UV-induced uncaging of glutamate in intact cells resulted in an increase in inter-subunit FRET and a decrease in intra-subunit FRET, which the authors believe represent movement of protomers towards each other and outward movement of TM6, respectively. Dimer rearrangement occurred with an average time constant of ~2ms, with 7TM conformational changes occurring approximately 10 times slower. Rapid solution exchange in outside-out Xenopus oocyte patches resulted in a similar two-step activation profile. Both methods revealed that initial mGlu1 dimer rearrangement occurs faster than previously reported [4,5], and is only loosely coupled to subsequent 7TM domain conformational changes. Receptor deactivation also occurred in two discrete steps, with inter-subunit rearrangements again preceding intra-subunit conformational changes. Occupancy of both binding sites was required for optimal activation and deactivation kinetics, as inter-subunit rearrangements in both directions were significantly slower in receptor mutants that only bind agonist in one protomer.
This study has revealed the existence of metastable intermediate activation states i.e. states in which the dimer rearrangement or the 7TM conformational changes exist in isolation. How these intermediate states influence mGlu1 signalling is unknown, as the fluorescently labelled mGlu1 dimers are unable to couple to G proteins [3]. Additionally, whether the intra-subunit FRET changes do indeed represent specific TM6 movements is somewhat ambiguous, given that the C-terminus to which CFP is attached is predicted to be highly flexible. However, should this activation mechanism be relevant and applicable across all Class C GPCRs, it may contribute to the complexity of Class C pharmacology. Allosteric agonists of Class C GPCRs bind to sites in the 7TM domain, activating receptors in the absence of orthosteric ligand [2], indicating that the 7TM-active state represents a physiologically relevant signalling conformation. These intermediate receptor activation states may also influence transducer coupling. Different orthosteric/allosteric ligand combinations shifting the balance between the various active states, stabilising unique conformations and engaging distinct downstream signalling pathways could play a part in the biased and probe dependent pharmacology apparent for many Class C GPCR ligands. Exploring multiple GPCRs with different orthosteric and allosteric ligand combinations is a crucial next step in understanding how the kinetics of receptor activation relates to ligand pharmacology. Understanding how drug-like compounds impact receptor activation kinetics and stabilise intermediate receptor states will likely play a large role in rational drug design programs going forward.
Comments by Shane D Hellyer (https://research.monash.edu/en/persons/shane-hellyer) and Karen J Gregory (https://research.monash.edu/en/persons/karen-gregory), Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Australia.
(1) Lohse M.J. et al. (2012). Fluorescence/bioluminescence resonance energy transfer techniques to study G protein-coupled receptor activation and signalling. Pharmacol Rev, 64: 299-336. [PMIDs: 22407612]
(2) Leach K & Gregory K.J. (2017) Molecular insights into allosteric modulation of Class C G protein-coupled receptors. Pharmacol Res, 116: 105-118. [PMIDs: 27965032]
(3) Hlacvackova V et al. (2012) Sequential inter- and intrasubunit rearrangements during activation of dimeric metabotropic glutamate receptor 1. Sci Signal, 5: ra59. [PMIDs: 22894836]
(4) Marcaggi P et al. (2009) Optical measurement of mGluR1 conformational changes reveals fast activation, slow deactivation, and sensitization. PNAS, 106: 11388-11393. [PMIDs: 19549872]
(5) Vafabakhsh R et al. (2015) Conformational dynamics of a class C G-protein-coupled receptor. Nature, 524: 497-501. [PMIDs: 26258295]
(6) Grushevskyi E.O. et al. (2019) Stepwise activation of a class C GPCR begins with millisecond dimer rearrangement. PNAS. pii: 201900261. doi:10.1073/pnas.1900261116. [Epub ahead of print] [PMIDs: 31023886]
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